Asparagine-linked glycosylation (N-glycosylation) is the most common co-translational modification found in eukaryotic proteins. As proteins are synthesized by the ribosome, the polypeptide enters the endoplasmic reticulum, where oligosaccharyl transferase (OT) attaches a branched carbohydrate (N-glycan) to the side chain of certain asparagine residues [Hirschberg, C. B., Snider, M. D. (1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 56, 63-87.] This process requires an Asn-X-Ser/Thr consensus sequence in the peptide substrate, where X is any amino acid except proline [Bause, E. (1983) Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331-6; Marshall, R. D. (1972) Glycoproteins. Annu Rev Biochem 41, 673-702.] After the attachment of the glycans, the carbohydrate moiety is extensively modified by a complex array of glycosidases and glycosyl transferases in the ER and golgi apparatus. The attached N-glycans are very important in protein folding, as well as directing the protein to the appropriate location within the cell [Dwek, R. A. (1996) Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev 96, 683-720; O'Connor, S. E., Imperiali, B. (1996) Modulation of protein structure and function by asparagine-linked glycosylation. Chem Biol 3, 803-12.] Outside the cell, the sugars aid in protein-protein interactions, often modulating the activity of the protein to which they are attached. Depending on the glycan composition, they can also protect against or facilitate protein degradation in circulation, as well as target the protein to a specific organ [Crocker, P. R., Varki, A. (2001) Siglecs in the immune system. Immunology 103, 137-45; Helenius, A., Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364-9; Imperiali, B., O'Connor, S. E. (1999) Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 3, 643-9.]
N-glycans also have an essential role in normal biology, as evidenced by the high lethality in cases of defective glycosylation. In mouse knockout models, disrupting even one of the biosynthetic enzymes can lead to enormous multisystemic disorders, and several result in embryonic lethality [Furukawa, K., Takamiya, K., Okada, M., Inoue, M., Fukumoto, S. (2001) Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes. Biochim Biophys Acta 1525, 1-12.] There are currently six recognized human congenital disorders of glycosylation (CDGs), all resulting in patients with multiple organ abnormalities, developmental delay and immune problems, among others [Jaeken, J., Matthijs, G. (2001) Congenital disorders of glycosylation. Annu Rev Genomics Hum Genet. 2, 129-51; Freeze, H. H., Aebi, M. (1999) Molecular basis of carbohydrate-deficient glycoprotein syndromes type I with normal phosphomannomutase activity. Biochim Biophys Acta 1455, 167-78; Carchon, H., Van Schaftingen, E., Matthijs, G., Jaeken, J. (1999) Carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase-deficiency). Biochim Biophys Acta 1455, 155-65.] In fact, the immune system is one of the most commonly studied systems where N-glycans have been shown to play an important physiological role. For example, specific carbohydrate structures are recognized by selectins, a family of proteins expressed on endothelial cells or lymphocytes that can trigger the immune system upon activation [Powell, L. D., Sgroi, D., Sjoberg, E. R., Stamenkovic, I., Varki, A. (1993) Natural ligands of the B cell adhesion molecule CD22 beta carry N-linked oligosaccharides with alpha-2,6-linked sialic acids that are required for recognition. J Biol Chem 268, 7019-27; Sgroi, D., Varki, A., Braesch-Andersen, S., Stamenkovic, I. (1993) CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. J Biol Chem 268, 7011-8.] The same class of structures that are necessary for proper immune function can also provide a binding site for certain viruses, bacteria or tumor cells in the body [Karlsson, K. A. (1998) Meaning and therapeutic potential of microbial recognition of host glycoconjugates. Mol Microbiol 29, 1-11; Pritchett, T. J., Brossmer, R., Rose, U., Paulson, J. C. (1987) Recognition of monovalent sialosides by influenza virus H3 hemagglutinin. Virology 160, 502-6.]
Viral infection is mediated by the interaction of viral proteins with N-glycans on the cell surfaces of the host [Van Eijk, M., White, M. R., Batenburg, J. J., Vaandrager, A. B., Van Golde, L. M., Haagsman, H. P., Hartshorn, K. L. (2003) Interactions of Influenza A virus with Sialic Acids present on Porcine Surfactant Protein D. Am J Respir Cell Mol. Biol.] Despite the increasing evidence associating glycans to different pathogenic conditions, in multiple instances it is unclear whether changes in N-glycan structure are a cause or a symptom of the disorder. In cystic fibrosis, increased antennary fucosylation (α1-3 linked to GlcNAc) is observed on surface membrane glycoproteins of airway epithelial cells [Glick, M. C., Kothari, V. A., Liu, A., Stoykova, L. I., Scanlin, T. F. (2001) Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells. Biochimie 83, 743-7; Scanlin, T. F., Glick, M. C. (2000) Terminal glycosylation and disease: influence on cancer and cystic fibrosis. Glycoconj J 17, 617-26.]
There have also been many reports of alterations in N-glycan composition on cancer cell proteins. For example, there are indications that prostate cancer cells produce prostate specific antigen (PSA) with more glycan branching than non-cancer cells [Peracaula, R., Tabares, G., Royle, L., Harvey, D. J., Dwek, R. A., Rudd, P. M., de Llorens, R. (2003) Altered glycosylation pattern allows the distinction between prostate-specific antigen. (PSA) from normal and tumor origins. Glycobiology 13, 457-70; Belanger, A., van Halbeek, H., Graves, H. C., Grandbois, K., Stamey, T. A., Huang, L., Poppe, I., Labrie, F. (1995) Molecular mass and carbohydrate structure of prostate specific antigen: studies for establishment of an international PSA standard. Prostate 27, 187-97; Prakash, S., Robbins, P. W. (2000) Glycotyping of prostate specific antigen. Glycobiology 10, 173-6.] Melanoma and bladder cancer cells produce proteins with highly branched glycans due to an overexpression of the biosynthetic enzyme β1,6-N-acetyl-glucosaminyltransferase V (GnT-V) [Chakraborty, A. K., Pawelek, J., Ikeda, Y., Miyoshi, E., Kolesnikova, N., Funasaka, Y., Ichihashi, M., Taniguchi, N. (2001) Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, beta1-6 branching, and metastasis. Cell Growth Differ 12, 623-30; Przybylo, M., Hoja-Lukowicz, D., Litynska, A., Laidler, P. (2002) Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines. Cancer Cell Int 2, 6.] Increased sialylation and additional branching have also been observed in cells from human breast and colon neoplasia [Lin, S., Kemmner, W., Grigull, S., Schlag, P. M. (2002) Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells. Exp Cell Res 276, 101-10; Nemoto-Sasaki, Y., Mitsuki, M., Morimoto-Tomita, M., Maeda, A., Tsuiji, M., Irimura, T. (2001) Correlation between the sialylation of cell surface Thomsen-Friedenreich antigen and the metastatic potential of colon carcinoma cells in a mouse model. Glycoconj J 18, 895-906; Dennis, J. W., Granovsky, M., Warren, C. E. (1999) Glycoprotein glycosylation and cancer progression. Biochim Biophys Acta 1473, 21-34; Fernandes, B., Sagman, U., Auger, M., Demetrio, M., Dennis, J. W. (1991) Beta 1-6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia. Cancer Res 51, 718-23.]